γ-L-glutamyl-L-cysteine ethyl ester and pharmaceutical compositions containing the same as an effective ingredient

ABSTRACT

γ-L-glutamyl-L-cysteine ethyl ester which has an activity to increase glutathione levels in tissues and a drug for treating liver diseases, cataracts, and kidney diseases containing it as the effective ingredient.

TECHNICAL FIELD

The present invention relates to γY-L-glutamyl-L-cysteine ethyl esterand agents to increase the tissue glutathione levels, medicines forliver diseases, drugs for cataract, and medicines for kidney diseases,which contain the same as an effective ingredient.

TECHNICAL BACKGROUND

Glutathione is present in almost all biological tissues as a majorintracellular reducing agent and plays important roles in catalysis,metabolism, transport and protection of cells. To be particular as tothe protection of cells, glutathione displays said function (1) byreductively destorying reactive oxygen compounds and free radicals withthe acid of glutathione peroxidase or (2) by reacting with intracellulartoxic compound with the aid of glutathione-S-transferase and thereaction product being excreted out of the cells as a glutathioneconjugate, thus playing roles in antioxidation, detoxication, protectionagainst radiation injury, and increasing resistance to temperature, etc.

Therefore, when the tissue content of glutathione has decreased due todiseases or aging, the tissue becomes liable to suffer injuries. It isimportant for the restoration of lost cellular functions to decreasedtissue glutathione levels to the normal value and it is also generallyregarded that the cellular protective function can be further enhancedby increasing the tissue glutathione even in case of a normal cell.Actually it has also been reported that glutathione and several kinds ofthiol compounds were used effective in protecting against mutagenic andcarcinogenic substances and further in reducing the tumor size of theanimal liver caused by from said malignant substances.

However, there are problems that half life of glutathione in blood isshort (several minutes) and administration of glutathione itself is notso useful in increasing tisse glutathione. It is considered that this isdue to the facts that glutathione itself can not be taken efficientlyinto cells, and exogenously supplied glutathione must be once degradedto its constituent peptide or amino acids, which are able to betransported and converted intracellularly to glutathione.

There have been several compounds that overcome problems above-mentionedand are proved to be superior to glutathione in increasing glutathionelevel of human lymphoma cells or animal tissues.2-oxothiazolidine-4-carboxylate, γ-L-glutamyl-L-cysteine, andr-L-glutamyl-L-cysteinyl-glycine ethyl ester (glutathione monoethylester), etc. (for instance, Curr. Top. Cell. Regul., vol. 26, pp383-394, 1985; Fed. Proc., vol. 43, pp 3031-3043, 1984).

Disclosure of the Invention

The present inventors have made a search for new compounds, which areeffective for increasing glutathione in the tissue, by using a primarycultured hepatoxyte and found that γ-L-glutamyl-L-cysteine ethyl esteris effective in increasing glutathione of the liver cells; thatespecially in case where glutathione level is decreased by use ofD,L-buthionine-sulfoximine, an inhibitor of glutathione synethesis, theaddition of said glutamyl compound after the removal of the inhibitormakes the glutathione level recover at a remarkably high speed; that theglutathione level recovering effect of this compound is much superior tothe compounds previously mentioned in the preceding paragraph; and thatthe compound prevents the necrosis of liver cells due to carbontetrachloride treatment, thus achieved the present invention.

More particularly, this invention relates to γ-L-glutamyl-L-cysteineethyl ester represented by the following formula ##STR1## and agents toincrease the tissue glutathione levels, medicines for liver diseases,drugs for cataract, and medicines for renal diseases, which contain thesame as an effective ingredient.

The aforementioned formula indicates compound of reduced form andoxidized form of γ-L-glutamyl-L-cysteine ethyl ester (oxidized typedimer), which is a dimer comprising a disulfide linkage (-S-S-) formedby the dehydrogenation reaction caused between the two molecules of thecompound of the aforementioned formula, is also included in the scope ofthis invention.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A, 1B and 1C are graphs showing how the compound of thisinvention acts to increase the tissue glutathione level uponadministration to mice.

FIGS. 2A and 2B are graphs showing how the compound of this inventionfunctions to mitigate the decrease of the tissue glutathione level whenit is administered to mice which have been pretreated with L-buthioninesulfoximine.

FIGS. 3A and 3B are graphs showing how the compound of this inventionfunctions with regard to tissue glutathione levels when administered tomice 1 day after administering L-buthionine sulfoximine.

BEST MODE OF CARRYING OUT THE INVENTION

The compound of this invention is a derivative of a compound which hashitherto been publicly known as γ-L-glutamyl-L-cysteine methyl ester andcan be synthesized organic chemically according to (1) a generally knownmethod such as one reported by Flohe et al. in Z. Klin. Chem. u. Klin.Biochem., vol. 8, pp 149-155 (1970). The compound can also besynthesized (2) by monoethyl esterifying γ-L-glutamyl-L-cysteine byadopting a method of monoethyl esterification of glutathione reported byAnderson et al. in Arch. Biochem. Biophys., vol. 239, pp 538-548 (1985).

The present compound has a function to increase the tissues glutathionelevels and is accordingly useful for the invigoration of various cellsand prevention of necrosis. In view of the assumption that (1) thedecrease of tissue glutathione level is the cause in part of adultdiseases such as cataract, hypertension, arterio sclerosis, diabetes,gastric ulcer, cancer, renal diseases respiratory diseases, and diseasesof brain and heart, the medicines of the present invention are effectivefor the prevention and therapy of the aforementioned diseases andfurther display marked curative effects as antitussive and expectorantagents, drugs for treating intoxication, various allergic diseases,cutaneous diseases, for preventing sunburn and radiation injury, and forincreasing temperature resistivity and (2) are furthermore effective forremedy and prevention of liver troubles such as hepatitis, fatty liver,and liver cirrhosis. They also (3) have a detoxifying action for therenal toxicity resulting from the use of an anti-cancer agent and aretherefore expected to be useful for the therapy and prevention of kidneydiseases.

Also, in view of the fact that some kinds of thiol compounds are usedfor treating various diseases including liver troubles, intoxication,eye complaints, tussis and obstructive phlegm because of theirantioxidative-reductive activity and action to bond with heavy metalsdue to their SH group, it may be reasonably expected that the presentcompounds, which have SH group, can display various remedial effects bythemselves or as metabolites.

The compounds of this invention can be administered orally orparenterally such as intravenously, intramuscularly, subcutaneously,reactally, percutaneously, and eye dropping. As the dosage form for oraladministration, tablet, pill, granule, powder, liquid preparation,suspension, and capsule may be mentioned.

As the method for preparing in the form of a tablet, it may be preparedaccording to the ordinary method in which excipients such as lactose,starch, and crystalline cellulose; binders such as carboxymethylcellulose, methyl cellulose, and polyvinyl pyrrolidone; anddisintegrators such as sodium alginate and sodium bicarbonate.

Pills, granules, and powders can also be prepared according to theordinary method by use of glyserin ester such as tricaprylin andtriacetin and alcohol such as ethanol. Capsules are prepared by fillinggelatin capsules with granules, powder, or liquid preparation.

As the dosage form for intravenous, intramuscular, and subcutaneousadministration, there are injections prepared in the form of an aqueousor nonaqueous solution. In preparing these injections, solvents such asisotonic sodium chloride solution, ethanol, and propylene glycol, and,if necessary, antiseptics and stabilizers are used.

For reactal administration, ordinary suppository made of gelatin softcapsule may be mentioned.

As the dosage form for percutaneous administration, ointments, forinstance, may be mentioned. They are prepared according to the ordinarymethod.

Eye drops can be prepared by use of an nearly neutral buffer solutioncomprising, for instance, sodium bicarbonate, sodium bisulfite, andboric acid, and, if necessary, antiseptics, stabilizers, and osmoticpressure regulators may also be used.

When the compound of this invention is used as the agent for increasingthe level of glutathione in the tissue, or remedy for treating liverdiseases, cataract, and kidney troubles, the dosage is usually about 2mg-20 g per day for an adult, though it varies depending upon thecondition of the patient. It may be given orally or parenterally bydivided administration ranging from 1 to 8 times a day.

The acute toxicity of the compound of this invention is very low ascompared with the control compounds as shown in Example 10.

The invention will be further illustrated by the following examples.

EXAMPLE 1 Synthesis and purification of γ-L-glutamyl-L-cysteine ethylester and its oxidized form

(i) Esterification

After 250 ml of hydrogen chloride-containing ethanol (7N HCl-EtOH) wasadded to a dehydrated ethanol suspension (750 ml) containing 50 g (about185 mmol) of γ-L-glutamyl-L-cysteine monohydrate, the mixture wasstirred at room temperature for 1 hour. Upon addition of hydrogenchloride, the suspension turned to a solution. The reaction solution wasthen poured into ether (7.5 l) which had been cooled to 0° C. beforehandand the mixture was kept stirring for 2 hours to make the crystalsprecipitate. Thus obtained product was separated by filtration to give48.4 g (83%) of γ-L-glutamyl-L-cysteine ethyl ester hydrochloride salt.

In the product thus obtained, 5-10% diester body is found existing. Inorder to obtain the desired compound in the form of a pure product, thehydrochloride slat should be neutralized with base such as amine andthen purified by use of an ion-exchange resin.

(ii) Purification

A solution prepared by dissolving 45 g of the aforementionedγ-L-glutanyl-L-cysteine ethyl ester hydrochloride salt in 950ml of waterwas neutralized with aqueous ammonia and furthermore water was added tomake the final volume of 1 l. The solution was made to pass continuouslythrough BIO-RAD's AG 50 W ammonium-form column (50 ml of resin) and AG:acetate-form column 125 ml of resin), (both of) which had beenwellwashed with water beforehand, and columes were finally washed withwater equivalent to the volume of the columns. The eluate and thewashings were put together and lyophilized to obtain 36 g ofγ-L-glutamyl-L-cysteine ethyl ester. Its physical properties are asfollows:

NMR (δppm, D₂ O): 1.28 (3H, t, J=7.0 Hz), 2.16 (2H, m), 2.54 (2H, t,J=7.6 Hz), 3.00 (2H, m), 3.79 (1H, t, J=6.3 Hz), 4.26 (2H, q, J=7.0 Hz),4.64 (1H, m)

When thus purified authentic sample was eluted on Waters' C₁₈ reversedphase column (4 mm×30 cm) at a flow rate of 1 ml/min, it showed thesingle peak of 4.7-minute retention time under the condition ofacetonitrile (15%)/0.1% trifluoroacetic acid (85%) and the single peakof 7.4-minute retention time under the condition of acetonitrile(7.5%)/trifluoroacetic acid (92.5%).

(iii) Preparation of the oxidized form

7 g of γ-L-glutamyl-L-cysteine ethyl ester obtained in the preceding(ii) was dissolved in 50 ml of water. The solution was adjusted to pH7.4with aqueous ammonia and after the addition of 10 mg of copper sulfatepentahydrate, air was blown thereinto overnight at room temperature.

The reaction solution was made to pass in succession through AG50W.ammonium form column 3 ml of resin) and AGl. acetate form column (3 mlof resin), which had been washed thoroughly with water, to remove coppersulfate therefrom and was lyophilized to obtain 6.30 g ofγ-L-glutamyl-L-cystein ethyl ester of oxidized type dimer. Its physicalproperties are shown below.

NMR (δppm, D₂ O): 1.4 (6H, t, J=7.0 Hz), 2.3 (4H, m), 2.55 (4H, m),3.0˜3.6 (4H, m), 3.9 (2H, m), 4.35 (4H, q, J=7.0 Hz), 4.9 (2H, m)

When this product was eluted on Shiseido's CAPCELL PAK C₁₈ reversedphase column (4.6 mm×25 cm) at a flow rate of 1 ml/min under the mixedsolvent condition of acetonitrile (20%)/0.1% trifluoroacetic acid (80%),the oxidized form eluated as single peak of 5.6-minute retention timeand the reducing type eluated as the single peak of 4.2-minute retentiontime under the same conditions.

EXAMPLE 2 Action to increase the glutathione levels in the liver cells

Hepatocyte (sample) was prepared according to the method of Nakamura etal. published in the Protein, Nucleic Acid, and Enzyme, vol. 24, pp55-76 (1981), partly adopting the method of Wang et al. reported in theIn Vitro, vol. 21, pp 526-530 (1985). The liver of mice was purfusedwith 0.05% collagenase, and cells were collected by centrifugation, andwere further centrifuged with percoll to collect the parenchymalhepatocytes. The obtained hepatocytes were dispersed in the Williams'medium E containing 10% bovine fetal serum, dexamethasone (10⁻⁶ M),insulin (1 μg/ml), and antibiotic, and were inoculated into the cultureplate at a density of 1×10⁵ cells/0.2 ml/cm². After unattached cellswere removed once 1.5 hours later, the cultivation was continued at 37°C. in an atomosphere of 5% CO₂ -95% air. The compound of this inventionwas added to the culture system to a concentration of 1 mM to measurethe time lapsed changes of the cellular glutathione level according tothe method reported by Owens and Belcher, Biochem. J., vol. 94, pp705-711 (1965) and Tietze, Anal. Biochem., vol. 27, pp 502-522 (1969).The similar measurements were also made without the addition of thecompound for comparison.

Table 1 shows time course change of the cellular glutathione level byrelative values in which the value of 100 represent the value of theexperiment before the addition of the compound.

                  TABLE 1                                                         ______________________________________                                                    Compound                                                                                     γ-L-glutamyl-L-                                            Control      cysteine ethyl ester                               Hours         (Not added)  (1 mM)                                             ______________________________________                                        0 (before addition)                                                                         100          100                                                0.5           100          140                                                1             100          170                                                2             100          200                                                ______________________________________                                    

To sum up the above table, it is apparent that the compound of thisinvention has an effect of increasing the glutathione levels in theliver cells.

EXAMPLE 3 Action to mitigate the decrease in glutathione levels in theliver cells when the compound of this invention is made to coexist withD,L-buthionine sulfoximine

The cultured liver cells were cultured for 13 hours after the additionof D,L-buthionine sulfoximine to the concentration of 0.3 mM and thepresent compound to the concentration of 1 mM and then the levels ofcellular glutathione were measured. The similar measurements were madeon the controls in which nothing was added and in which D,L-buthioninesulfoximine only was added.

Table 2 shows the celular glutathione levels by relative values based onthe value of 100 before the addition of the compounds.

                  TABLE 2                                                         ______________________________________                                                           0 hour                                                                        (before  13 hours                                                             addition)                                                                              later                                             ______________________________________                                        Control (nothing added)      100      100                                     D,L-buthionine sulfoximine                                                                    0.3   mM     100      17                                      Coexistence of:                                                               D,L-buthionine sulfoximine                                                                    0.3   mM     100      30                                      γ-L-glutamyl-L-cysteine                                                                 l     mM                                                      ethyl ester                                                                   ______________________________________                                    

It is apparent from the above table that the present compound has aneffect to mitigate the decrease of the glutathione levels in the livercells due to D,L-buthionine sulfoximine.

EXAMPLE 4 Action to promote the recovery of cellular glutathione levelsand action to prevent the necrosis of liver cells resulting from the useof the present product in case where the cellular glutathione level isonce decreased by use of D,L-buthionine sulfoximine and the inhibitor isremoved thereafter.

D, L-buthionine sulfoximine was added to the cultured liver cells to adensity of 0.5 mM and cultivation was carried on for 16 hours.D,L-buthionine sulfoximine was then removed and the compound of thisinvention and control compounds (equimolar mixture of glutamic acid,cysteine, and glycine; glutathione; glutathione monoethyl ester;γ-L-glutamyl-L-cysteine) were respectively added to each culture to adensity of 1 mM and was followed by 2-hour cultivation. The changes ofcellular glutathione levels were measured at that time. Thereafter thecompounds studied were removed from the respective cultures whoseculture media were then replaced with new ones containing carbontetrachloride at a density of 0.5 μl/ml. After 1-hour cultivation, theaction to prevent the necrosis of liver cells was examined by measuringthe GPT activity of the respective culture supernatants and Trypan Bluestaining test.

In Table 3, the changes of cellular glutathione levels are expressed byrelative values based on the untreated value set at 100 and the actionto prevent the necrosis of liver cells is indicated by the GPT activityof the culture supernatant and survival rate.

                                      TABLE 3                                     __________________________________________________________________________                               GPT activity                                                            Cellular                                                                            of culture                                                                           Survival                                                         glutathion                                                                          supernatant                                                                          rate                                                             level (until/ml)                                                                           (%)                                         __________________________________________________________________________    Control (not treated)                                                                              100   3      100                                         D,L-buthionine sulfoximine                                                                         1     --     --                                          0.5 mM (16 hours)                                                             Liquid culture medium only, after the                                                              1.5   80     2                                           removal of D,L-buthionine sulfoximine                                         (2 hours)                                                                     Mixture of glutamic acid, cysteine, and                                                            5.5   70     5                                           glycine each 1 mM added                                                       Glutathione 1 mM added                                                                             10    60     10                                          Glutathione monoethyl ester 1 mM added                                                             15    35     25                                          γ-L-glutamyl-L-cysteine 1 mM added                                                           10    60     10                                          γ-L-glutamyl-L-cysteine ethyl ester                                                          50    3      100                                         (of this invention) 1 mM added                                                __________________________________________________________________________

It is now apparent that γ-L-glutamyl-L-cysteine ethyl ester can recoverthe glutathione level of liver cells once decreased by D,l-buthioninesulfoximine much faster than the cases where the compound of thisinvention is not added or other control compounds mentioned hereinbeforeare added and also displays the most excellent effect to prevent thenecrosis of liver cells. It is, therefore, expected that the compound ofthis invention is effective for the therapy and prevention of liverdiseases such as hepatitis, fatty liver, and liver cirrhosis.

EXAMPLE 5 Action to recover the glutathione levels of liver cells uponthe removal of carbon tetrachloride and addition of the present compoundafter the cellular glutathione level was once decreased by carbontetrachloride

After carbon tetrachloride was added to the cultured liver cells at aconcentration of 0.5 μl/ml, culture was continued for 1 hour. Theculture had then its medium exchanged with a new liquid culture mediumand also had the present compound added at a density of 1 mM. One hourlater, the change of the cellular glutathione level was measured.

Table 4 shows the changes of the cellular glutathione level in terms ofrelative values based on the value of 100 before the carbontetrachloride treatment.

                  TABLE 4                                                         ______________________________________                                                                 Cellular                                                                      glutathione                                                                   level                                                ______________________________________                                        Control (not treated)          100                                            After treated with carbon tetrachlodie                                                                       33                                                                  (0.5 μl/ml)                                           No compound added after the removal                                                                          13                                             of carbon tetrachloride                                                       Addition of γ-L-glutamyl-L-cysteine                                                                    67                                             ethyl ester after the removal of carbon                                       tetrachloride,       l mM                                                     ______________________________________                                    

The present compound has an effect to recover the cellular glutathionelevels which have been decreased by carbon tetrachloride.

EXAMPLE 6 Action to increase the tissue glutathione levels in case wherethe present compound is given to mice

ICR male mice (9-day old) were subcutaneously given 2.5 mmol/kg of thecompound of this invention (r-Glu-Cys OET) or control compounds(glutathione (GSH), γ-L-glutamyl-L-cysteine (r-Glu-Cys)) four times at2.5-hour intervals. The mice were dissected and their livers and kidneyswere removed before the administration and every 2.5 hour after theadministration to have their glutathione (GSH) level determined. As foreyeballs, besides the determination of glutathione contents, the SHcontents were also determined by the DTNB method upon administration of5 mmol/kg of the present compound after the same procedure. The resultsare shown in FIGS. 1A, 1B and 1C.

It is clearly shown in FIGS. 1A, 1B and 1C that the compound of thisinvention has far more excellent effect than glutathione andγ-L-glutamyl-L-cysteine in increasing the tissue glutathione levels ofthe liver (FIG. 1 (A)) and kidney (FIG. 1 (B)) even in case of animals.It is also seen that the compound has also an effect to improve the SHlevels remarkably in addition to the aforementioned effect in case ofeyeballs (FIG. 1 (C)).

EXAMPLE 7 Action to mitigate the decrease of tissue glutathione levelsin case where the present compound and L-buthionine sulfoximine arealternately given to mice

The compound of this invention was administered in the same way asExample 6, and L-buthionine sulfoximine (BSO) was administered likewisein dosage of 4 mmol/kg one hour after every administration of saidcompound. The contents of glutathione in the livers and kedneys weremeasured according to the methods described in Example 6 every 2.5 hoursafter the initial administration of the present compound. As controls,saline was administered in place of the present compound to take thesimilar measurements. FIGS. 2A and 2B shown the results.

It has been made apparent that the present compound has an effect tomitigate the decrease of tissue glutathione levels of the liver (FIG. 2Aand kidney induced by L-buthionine sulfoximine, even in case of animalmodels (FIG. 2 (B)).

EXAMPLE 8 Action to promote the recovery of tissue glutathione levels incase where the present compound is administered to mice pretreated withL-buthionine sulfoximine

ICR male mice (9-day old) were given L-buthionine sulfoximine in dosageof 4 mmol/kg four times at an interval of 2.5 hours to decrease theirtissue glutathione levels beforehand. The next day, the mice were givensubcutaneously 2.5 mmol/kg of the present compound or saline 4 times atan interval of 2.5 hours. Glutathione monoethyl ester (GSHOET) was alsogiven likewise in dosage of 2.5 mmol/kg as a control. The contents ofglutathione in the livers and kidneys were measured according to themethods described in Example 6. The results are shown in FIGS. 3A and3B.

The present compound showed the same effectiveness as glutathionemonoethyl ester in recovering the tissue glutathione levels of the liver(FIG. 3 (A)). With regard to the kidney (FIG. 3B, however, the presentcompound showed far more excellent effect in making the tissularglutathion level recover to the normal level as compared withglutathione monoethyl ester.

It is clear that, in cases of animal models too, the present compoundhas far more excellent effect of quickly recovering the tissueglutathione levels decreased by L-buthionine sulfoximine as compared toglutathione monoethyl ester, not to speak of a case where no treatmentwas made.

EXAMPLE 9 Protective effect of the present compounds (of reduced formand oxidized form) on experimentally developed cataract induced byL-buthionine sulfoximine

Experimental cataract was prepared according to the method of Calvin etal., Science, vol. 233, pp 553-555 (1986). More particularly,L-buthionine sulfoximine was administered subcutaneously to the ICR malemice at a dose of 4 mmol/kg for 3 successive days from the 9th day tothe 11th day after birth every 2.5 hours 4 times a day to causecataract. The rate of cataractgenesis was determined on the 16th dayafter birth. The test compounds were also given subcutaneously for 3days from the 9th day to the 11th day after birth 4 times a day 1 hourbefore the administration of L-buthionine sulfoximine and thereafter thetest compounds studied only were subcutaneously given 4 times a day atan interval of 2.5 hours on the consecutive 12th and 13th days afterbirth and the protective effect of the test compound on cataractformation was examined on the 16th day after birth. As the controlcompounds, glutathione, glutathione monoethyl ester, andγ-L-glutamyl-L-cysteine were also examined.

Table 5 shows protective effect of the respective compounds studied oncataract formation.

The cataract development rate in the table means the ratio of theexperimental animals developing cataract to all the animals examined (11to 12 mice per group) and the cataractous eye rate means the ratio ofcataractous eyes to the whole number of eyes examined.

                  TABLE 5                                                         ______________________________________                                                      Dose of   Cataract                                                            compound  development                                                                              Cataractous                                              studied   rate       eye rate                                   Treatment     (mmol/kg) (%)        (%)                                        ______________________________________                                        Not treated   --        0          0                                          L-buthionine sulfoximine                                                                    --        91.7       91.7                                       (4 mmol/kg)                                                                   L-buthionine sulfoximine                                                                    1.25      75.0       70.8                                       + glutathione                                                                 L-buthionine sulfoximine                                                                    2.25      50.0       29.2                                       + glutathione                                                                 monoethly ester                                                               L-buthionine sulfoximine                                                                    1.25      50.0       41.7                                       + glutathione                                                                 monoethyl ester                                                               L-buthionine sulfoximine                                                                    1.25      66.7       77.8                                       L-glutamyl-L-                                                                 cysteine                                                                      L-buthionine sulfoximine                                                                    2.50      0          0                                          L-glutamyl-L-                                                                 cysteine ethyl ester                                                          (compound of                                                                  this invention)                                                               L-buthionine sulfoximine                                                                    1.25      50.0       41.7                                       L-glutamyl-L-                                                                 cysteine ethyl ester                                                          (compound of                                                                  this invention)                                                               L-buthionine sulfoximine                                                                    2.50      30.0       35.0                                       + oxidized form γ-L-                                                    glutamyl-L-cysteine                                                           ethyl ester                                                                   (compound of                                                                  this invention)                                                               ______________________________________                                    

If is clear from the above table that the compounds of this invention(inducing reduced form and oxidized form) have a strong protectiveactivity against experimental cataract induced by L-buthioninesulfoximine.

EXAMPLE 10 Acute toxicity of the present compound (mouse newborns)

Acute toxicity of the present compound was examined where it isadministered according to the schedule of Example 9 and compared withthere of controls, glutathione, glutathione monoethyl ester, andγ-L-glutamyl-L-cysteine. Table 6 shows the surviving rates of theanimals after the ending of administration of the compounds studied.

When the present compound was given to the animals at a dosage of 2.5mmol/kg, all the experimental animals survived, which shows that thecompound is as almost equally safe as glutathione monoethyl ester andapparently has much more safety than glutathion orγ-L-glutamyl-L-cysteine. Also, the present compound showed an effect tomitegate suppressed increment of animals' body weight by the toxicity ofBSO though such effect was not seen with glutathione orr-L-glutamyl-L-cysteine. It is, therefore, expected that, when thepresent compound is used in the treatment of cataract, it has highsafety as compared with the control compounds.

                  TABLE 6                                                         ______________________________________                                                                 Surviving                                            Compound                 rate (%)                                             ______________________________________                                        L-buthionine sulfoximine (4 mmol/kg)                                                                   100                                                  L-buthionine sulfoximine +                                                                              50                                                  glutathione (2.5 mmol/kg)                                                     L-buthionine sulfoximine +                                                                             100                                                  glutathione (1.25 mmol/kg)                                                    L-buthionine sulfoximine +                                                                             100                                                  glutathione monoethyl ester (2.5 mmol/kg)                                     L-buthionine sulfoximine +                                                                             100                                                  glutathione monoethyl ester (1.25 mmol/kg)                                    L-buthionine sulfoximine +                                                                              0                                                   γ-L-glutamyl-L-cysteine (2.5 mmol/kg)                                   L-buthionine sulfoximine +                                                                              75                                                  γ-L-glutamyl-L-cysteine (1.25 mmol/kg)                                  L-buthionine sulfoximine +                                                                             100                                                  γ-L-glutamyl-L-cysteine ethyl ester                                     (2.5 mmol/kg) (of this invention)                                             L-buthionine sulfoximine +                                                                             100                                                  γ-L-glutamyl-L-cysteine ethyl ester                                     (1.25 mmol/kg) (of this invention)                                            L-buthionine sulfoximine + oxidized form                                                               100                                                  γ-L-glutamyl-L-cysteine ethyl ester                                     (2.5 mmol/kg) (of this invention)                                             ______________________________________                                    

EXAMPLE 11 Action to detoxify renal toxicity induced by cisplatin

Cisplatin was administered intraperitoneally to ICR male mice (5-weekold) at a dose of 20 mg/kg. The present compound (2.5 mmol/kg) wasintraperitoneally given to the mice at 2.5-hour intervals 4 times a dayfor 5 consecutive days including the day on which cisplatin wasadministered. The control groups were given saline instead of thepresent compound. The blood was collected one week after cisplatinadministration to determine urea nitrogen levels in the blood serum(BUN). The result is shown in Table 7 (mean value of 10 mice per group).The present compound detoxified the renal disturbance activity ofcisplatin seen with the increase of BUN.

                  TABLE 7                                                         ______________________________________                                                               BUN                                                    Treatment              (mg/dl)                                                ______________________________________                                        Not treated (normal value)                                                                           20.8                                                   Cisplatin + saline     49.0                                                   L-glutamyl-L-cysteine  35.7                                                   ethyl ester                                                                   ______________________________________                                    

We claim:
 1. γ-L-glutamyl-L-cysteine ethyl ester expressed by thefollowing formula or its dimeric oxidized form: ##STR2## wherein saiddimeric oxidized form is formed by dehydrogenation between two moleculesof said γ-L-glutamyl-L-cysteine ethyl ester.
 2. A pharmaceuticalcomposition to increase tissue glutathione levels comprisingγ-L-glutamyl-L-cysteine ethyl ester or its dimeric oxidized form as theeffective ingredient, wherein said dimeric oxidized form is formed bydehydrogenation between two molecules of said γ-L-glutamyl-L-cysteineethyl ester, in a pharmaceutically acceptable carrier.
 3. A hepatotoniccomposition containing γ-L-glutamyl-L-cysteine ethyl ester or itsdimeric oxidized form as the effective ingredient, wherein said dimericoxidized from is formed by dehydrogenation between two molecules of saidγ-L-glutamyl-L-cysteine ethyl ester, in a pharmaceutically acceptablecarrier.
 4. A pharmaceutical composition for treating cataractscontaining γ-L-glutamyl-L-cysteine ethyl ester or its dimeric oxidizedform as the effective ingredient, wherein said dimeric oxidized form isformed by dehydrogenation between two molecules of said γ-L-glutamylL-cysteine ethyl ester, in a pharmaceutically acceptable carrier.
 5. Apharmaceutical composition for treating kidney diseases containingγ-L-glutamyl-L-cysteine ethyl ester or its dimeric oxidized form as theeffective ingredient, wherein said dimeric oxidized form is formed bydehydrogenation between two molecules of said γ-L-glutamyl-L-cysteineethyl ester, in a pharmaceutically acceptable carrier.